Summary- Significant progress was made on this project, which involves the development of medications for psychiatric diseases. Four papers were published in peer-reviewed journals. In one notable article, we report that increasing the size of the N-alkyl substituent on the compound 3,4-methylenedioxyamphetamine converts the drug from a transporter releaser into a transporter blocker. These finding have ramifications for designing new medications which target transporters. Specifically, the results demonstrate that increasing steric bulk on the amine group of amphetamine-type compounds will render the drugs less apt to serve as substrate-type releasers, thereby reducing their tendency to produce adverse effects in monoaminergic cells. Determining the structural elements that define substrates and inhibitors at the monoamine transporters is critical to elucidating mechanisms underlying these disparate functions. In this study, we addressed this question directly by generating a series of N-substituted-3,4-methylenedioxyamphetamine (MDA) analogs that differ only in the number of methyl substituents on the terminal amine group. Starting with 3,4-methylenedioxy-Nmethylamphetamine (MDMA), 3,4-ethylenedioxy-N,N-dimethylamphetamine (MDDMA) and 3,4-methylenedioxy-N,N,N-trimethylamphetamine (MDTMA) were prepared. We evaluated functional activities of the compounds at all three monoamine transporters in native brain tissue and in cells expressing the transporters. In addition, we used ligand docking to generate models of the respective protein-ligand complexes, which allowed us to relate experimental findings to available structural information. Our results suggest that the 3,4-methylenedioxy amphetamine analogs bind at the monoamine transporter orthosteric binding site by adopting one of two mutually exclusive binding modes: MDA and MDMA adopt a high-affinity binding mode consistent with a transportable substrate, whereas MDDMA and MDTMA adopt a low-affinity binding mode consistent with an inhibitor, in which the ligand orientation is inverted. Importantly, MDDMA can alternate between both binding modes while MDTMA exclusively binds to the low-affinity mode. Our experimental results are consistent with the idea that the initial orientation of bound ligands is critical for subsequent interactions that lead to transporter conformational changes and substrate translocation.